The goal of this proposal is the identification of all signaling pathways, and at least some components of each, regulated by Arf proteins in eukaryotes. This should include both unexpected and unproven pathways of Arf signaling. Each of the approaches described is designed to take advantage of the documented, conserved functions of Arf proteins throughout eukaryotic evolution to provide novel insights into the mechanisms and diversity of actions of this essential family of regulatory proteins. This proposal has four specific aims. (1) Identify genes which are synthetically lethal with art1 - as a result of either mutation or deletion. This screen will use unknown synthetic lethals (ARF2, SEC7, SEC21 and YPT1) to monitor saturation of the genome. Note that this will only identify those genes with genetic interactions with arf1 - in its essential role in vegetative growth, and not sporulation or other activities. (2) Screen for and analyze high copy suppressors of loss-or-Arf-function mutants, including: (A) suppressors of temperature sensitivity of fluoride supersensitivity of arf1-2, (B) suppressors of temperature sensitivity of arfl-3, and C suppressors of cold sensitivity of arfl-26. (3) Clone suppressors of the sporulation defect associated with mutations in ARF1. This may prove to be the most direct test of the hypothesis that Arf proteins regulate distinct signaling pathways in yeast through protein interactions with distinct molecular components. (4) Clone a human Arf GTPase activating protein (GAP). This is the most 'directed' goal in the application as it will attempt to clone a specific gene with a predetermined encoded activity.